Bottom Line:
Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level.Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells.Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins.

ABSTRACTMechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.

Mentions:
We used chicken embryos at their 18th day of incubation for isolation of hair cells, non-sensory and supporting cells. We focused on the vestibular maculae of the utricle and saccule for three reasons: i) they comprise the largest hair cell-bearing organs of the inner ear containing more than 20,000 hair cells per utricular macula, ii) the hair cells are functional at this late embryonic age [15], and iii) utricles and saccules can be dissected relatively quickly in larger numbers. After dissection and removal of otolithic membranes, the tissues were exposed to the styryl dye AM1-43 or FM1-43FX (Fig. 1A,D). Brief exposure to either of these dyes intensely labels living hair cells, whereas supporting and other non-sensory cells remain unlabeled [13] (Fig. 1C,F). Differentially labeling of hair cells and non-sensory cells is the basis for subsequent separation of hair cells from residual unlabeled cells of the sensory epithelia by flow cytometry. Specificity of the dye-uptake was confirmed by immunocytochemistry with antibodies to the hair cell marker myosin VIIA (Fig. 1C, F). After hair cell labeling, the sensory epithelia of the utricles and saccules were enzymatically detached from underlying stromal cells, mechanically separated from the stromal layer, and the living epithelia consisting of labeled hair cells and unlabeled non-sensory cells including supporting cells were collected in fresh media (Fig. 1B, E).

Mentions:
We used chicken embryos at their 18th day of incubation for isolation of hair cells, non-sensory and supporting cells. We focused on the vestibular maculae of the utricle and saccule for three reasons: i) they comprise the largest hair cell-bearing organs of the inner ear containing more than 20,000 hair cells per utricular macula, ii) the hair cells are functional at this late embryonic age [15], and iii) utricles and saccules can be dissected relatively quickly in larger numbers. After dissection and removal of otolithic membranes, the tissues were exposed to the styryl dye AM1-43 or FM1-43FX (Fig. 1A,D). Brief exposure to either of these dyes intensely labels living hair cells, whereas supporting and other non-sensory cells remain unlabeled [13] (Fig. 1C,F). Differentially labeling of hair cells and non-sensory cells is the basis for subsequent separation of hair cells from residual unlabeled cells of the sensory epithelia by flow cytometry. Specificity of the dye-uptake was confirmed by immunocytochemistry with antibodies to the hair cell marker myosin VIIA (Fig. 1C, F). After hair cell labeling, the sensory epithelia of the utricles and saccules were enzymatically detached from underlying stromal cells, mechanically separated from the stromal layer, and the living epithelia consisting of labeled hair cells and unlabeled non-sensory cells including supporting cells were collected in fresh media (Fig. 1B, E).

Bottom Line:
Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level.Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells.Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins.

ABSTRACTMechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.